RIDA GENE Bacterial Stool Panel I real-time PCR

Transcription

1 RIDA GENE Bacterial Stool Panel I realtime PCR Art. No.: PG Reactions For in vitro diagnostic use. 20 C RBiopharm AG, An der neuen Bergstraße 17, D64297 Darmstadt, Germany Tel.: +49 (0) / Telefax: +49 (0)

2 1. Intended use For in vitro diagnostic use. RIDA GENE Bacterial Stool Panel I is a multiplex realtime PCR for the direct, qualitative detection and differentiation of STEC, Salmonella spp., Campylobacter spp. and EIEC/Shigella spp. in human stool samples. RIDA GENE Bacterial Stool Panel I multiplex realtime PCR is intended for use as an aid in diagnosis of gastrointestinal infections caused by bacteria. 2. Summary and Explanation of the test Diarrheal disease is a major health care problem and causes about 2 billion cases worldwide. The World Health Organization (WHO) ranks diarrheal disease as 2nd most common cause of child deaths among children under 5 years globally, particularly in developing countries. About 1.9 million children younger than 5 years of age perish from diarrhea each year, more than AIDS, malaria and measles combined. 1,2 Common causes of bacterial diarrheal disease are Campylobacter spp., Salmonella spp. and Y. enterocolitica. Campylobacter species are one of the most common causes of bacterial diarrhea worldwide, responsible for 400 million 500 million cases annually. The disease caused by the genus Campylobacter is called campylobacteriosis. More than 80% of Campylobacter infections are caused by C. jejuni. The Centers for Disease Control and Prevention (CDC) estimates more than 2 million cases of campylobacteriosis each year in the US. The Foodborne Diseases Active Surveillance Network (FoodNet) reported an incidence rate of 13 cases per 100,000 population in C. jejuni was detected in 5 16% of children with diarrhea in developed countries and in 8 45% of children with diarrhea in developing countries. 4 Approximately 100 persons with Campylobacter infections die each year in the US. 3,4 Infection with Campylobacter occurs through contaminated food, especially poultry, water, contact with infected animals or by fecaloral route, particularly in children. The infectious dose is with 500 bacteria relatively low. After an incubation period of 2 to 5 days people with campylobacteriosis get fever, diarrhea, abdominal cramps, vomiting, abdominal pain and nausea. Potential longterm complications are autoimmune disorders, for example the GuillainBarré syndrome (GBS). 4 Salmonella species are also a leading cause of bacterial gastroenteritis worldwide. The genus Salmonella is divided into two species, S. enterica and S. bongori. So far, more than 2,500 Salmonella serotpyes are described which are pathogenic for humans. Salmonella species are causing nontyphoidal salmonellosis or typhoid fever. It is estimated that 93.8 million cases of nontyphoidal salmonellosis infections with 2 RIDA GENE Bacterial Stool Panel I

3 155,000 deaths occuring globally each year. 6 The CDC estimates more than 1.2 million cased of nontyphoidal salmonellosis infections each year in the United States, with more than 23,000 hospitalizations and 450 deaths. 5 Most of the nontyphoidal salmonellosis infections are caused by the S. typhimurium and S. enteritidis, while typhoid fever is caused by S. typhi and S. paratyphi A, B or C. The CDC estimates more than 1,800 cases of typhoid fever annually in the U.S. Transmission of Salmonella occurs through contaminated food, water or contact with infected animals. The infectious dose of Salmonella species is varying from 1 to 1000 bacteria. Nontyphoidal salmonellosis infection occurs after an incubation period of 6 72h with clinical symptoms of nausea, vomiting, abdominal cramps, diarrhea, fever and headache. People with typhoid fever get headache, achiness, high fever (from 39 C to 41 C), gastrointestinal symptoms, including abdominal pains and diarrhea within 1 to 3 weeks after exposure to the organism. 3,7 One of the six well known human pathogenic E. coli is the enteroinvasive E. coli (EIEC). In developing countries and also in returning travelers from those countries, EIEC can lead to shigelloselike infections since they are biochemically and genetically closely related to Shigella spp. The pathogenicity of EIEC and also Shigella spp. depends on the plasmiddependent ability to invade colonic epithelial cells and destroy them. By detection of the ipah gene (invasion plasmid antigen H gene) EIEC/Shigella spp. can be differentiated from ETEC. Clinical symptoms of EIECcaused Shigellosis are characterized by continuous abdominal cramps with watery and sometimes bloody diarrhea. Sources of infection are primarily contaminated water and food, but transmission from human to human is possible. Besides EIEC/Shigella spp., also the enterohaemorrhagic E. coli play an important role. Every year, approxiamtely 1000 EHEC infections are reported in Germany. EHEC are a subgroup of the shigatoxin producing E. coli (STEC or VTEC) and have the ability to produce two cytotoxins, verotoxin 1 and 2. Due to the close similarity of the verotoxins to the shiga toxins of Shigella dysenteriae, the VTEC are also called STEC. The classic laboratory diagnostic method of bacterial gastrointestinal infections is culture which takes several days. RIDA GENE Bacterial Stool Panel I multiplex realtime PCR is an attractive alternative method for testing stool samples and has proven to be highly sensitive and specific for the simultaneous detection of four of the most important diarrhea causing bacteria (Campylobacter spp., Salmonella spp. and EIEC/Shigella spp.). RIDA GENE Bacterial Stool Panel I

4 3. Test principle The RIDA GENE Bacterial Stool Panel I assay is a multiplex realtime PCR for the direct, qualitative detection and differentiation of Salmonella spp., Campylobacter spp. and EIEC/Shigella spp. After DNA isolation, amplification of the gene fragments specific for STEC (stx1/stx2), Salmonella spp. (ttr), Campylobacter spp. (16srRNA) and EIEC/Shigella spp. (ipah) occurs, if present. The amplified targets are detected with hydrolysis probes, which are labeled at one end with a quencher and at the other end with a fluorescent reporter dye (fluorophore). In the presence of a target the probes hybridize to the amplicons. During the extension step the Taqpolymerase breaks the reporterquencher proximity. The reporter emits a fluorescent signal which is detected by the optical unit of a realtime PCR instrument. The fluorescence signal increases with the amount of formed amplicons. The RIDA GENE Bacterial Stool Panel I assay contains an Internal Control DNA (ICD) as an internal control of sample preparation procedure and to determine possible PCRinhibition. 4. Reagents provided Tab. 1: Reagents provided (Reagents provided in the kit are sufficient for 100 determinations) Kit Code Reagent Amount Lid Color 1 Reaction Mix 2x 1100 µl yellow 2 TaqPolymerase 1x 11 µl red D Internal Control DNA 2x 1800 µl orange N PCR Water 1x 500 µl white P Positive Control 1x 200 µl blue 5. Storage instructions Protect all reagents from light and store at 20 C. All reagents can be used until the expiration date. After expiry the quality guarantee is no longer valid. Carefully thaw reagents before using (e.g. in a refrigerator at 2 8 C). Reagents can sustain up to 5 freeze/thaw cycles without influencing the assay performance (e.g. after the first thawing separate it in aliquots and freeze immediately). During PCR preparation all the reagents should be stored cold in an appropriate way (2 8 C). 4 RIDA GENE Bacterial Stool Panel I

5 6. Additional necessary reagents and necessary equipment The RIDA GENE Bacterial Stool Panel I realtime PCR Assay is suitable for use with following extraction platforms and realtime PCR instruments: Extraction platform: RIDA Xtract (RBiopharm) Maxwell 16 (Promega) Realtime PCR instrument: Roche: LightCycler 480II Agilent Technologies: Mx3005P (with ATTOFilter) If you want to use other extraction platforms or realtime PCR instruments please contact RBiopharm at mdx@rbiopharm.de. RIDA GENE Color Compensation Kit IV (PG0004) for use with the LightCycler 480II Realtime PCR consumables (plates, tubes, foil) Centrifuge with a rotor for the reaction vials Vortexer Pipettes ( µl, µl, µl) Filter tips Powderfree disposal gloves RIDA GENE Bacterial Stool Panel I

6 7. Precautions for users For invitro diagnostic use. This test must only be carried out by trained laboratory personnel. The guidelines for working in medical laboratories have to be followed. The instruction manual for the test procedure has to be followed. Do not pipet samples or reagents by mouth. Avoid contact with bruised skin or mucosal membranes. During handling reagents or samples, wear appropriate safety clothing (appropriate gloves, lab coat, safety goggles) and wash your hands after finishing the test procedure. Do not smoke, eat or drink in areas where samples or reagents are being used. Extraction, PCR preparation and the PCR run should be separated in different rooms to avoid crosscontaminations. Samples must be treated as potentially infectious as well as all reagents and materials being exposed to the samples and have to be handled according to the national safety regulations. Do not use the kit after the expiration date. All reagents and materials used have to be disposed properly after use. Please refer to the relevant national regulations for disposal. For more details see Material Safety Data Sheets (MSDS) at 8. Collection and Storage of Samples 8.1 DNA isolation from Stool samples For DNA isolation of human stool samples, use a commercially available DNA isolation kit (e.g. RIDA Xtract) or DNA extraction system (e.g. Maxwell 16 (Promega)). Extract DNA according to the manufacturer s instructions. We recommend to dilute the stool samples before extraction 1:3 with water. Vortex the diluted stool sample intensely and centrifuge at 1000 x g for 30 sec. From the supernatant use the appropriate volume according to the manufacturer s instructions. The RIDA GENE Bacterial Stool Panel I realtime PCR kit contains an internal control (ICD) that detects PCR inhibition, monitors reagent integrity and confirms that nucleic acid extraction was sufficient. 6 RIDA GENE Bacterial Stool Panel I

7 If the ICD is used only as a PCR inhibition control, 1 µl of the ICD should be added to the MasterMix (see Tab. 3). If the ICD is used as an extraction control for the sample preparation procedure and as PCR inhibition control, 20 µl of the ICD has to be added during extraction procedure. The ICD should always be added to the specimenlysis buffer mixture and not directly to the specimen. 9. Test procedure 9.1 MasterMix preparation Calculate the total number of PCR reactions (sample and control reactions) needed. One positive control and negative control must be included in each assay run. We recommend to calculate an additional volume of 10 % to compensate imprecise pipetting (see Tab. 2, Tab. 3). Thaw, mix gently and centrifuge briefly the Reaction Mix, the TaqPolymerase, the Positive Control, the PCR Water and the Internal Control DNA before using. Keep reagents appropriately cold during working step (2 8 C). Tab. 2: Calculation and pipetting example for 10 reactions of the MasterMix (ICD as extraction and PCR inhibition control) Kit code MasterMix components Volume per reaction 10 reactions (10 % extra) 1 Reaction Mix 19.9 µl µl 2 TaqPolymerase 0.1 µl 1.1 µl Total 20.0 µl 220 µl Mix the components of the MasterMix gently and briefly spin down. Tab. 3: Calculation and pipetting example for 10 reactions of the MasterMix (ICD only as PCR inhibition control) Kit Code MasterMix components Volume per reaction 10 reactions (10 % extra) 1 Reaction Mix 19.9 µl µl 2 TaqPolymerase 0.1 µl 1.1 µl D Internal Control DNA 1.0 µl 11 µl Total 21.0 µl µl Mix the components of the MasterMix gently and briefly spin down. RIDA GENE Bacterial Stool Panel I

8 9.2 Preparation of the PCRMix Pipette 20 µl of the MasterMix in each reaction vial (tube or plate). Negative control: Add 5 µl PCR Water as negative control to the prepipetted MasterMix. Note: If the ICD is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICD to the negative control PCRMix. Sample: Add 5 µl DNAExtract to the prepipetted MasterMix. Positive control: Add 5 µl Positive Control to the prepipetted MasterMix. Note: If the ICD is used as extraction control for the sample preparation procedure and as PCR inhibition control, we recommend to add 1 µl of the ICD to the positive control PCRMix. Cover tubes or plate. Spin down and place in the realtime PCR instrument. The PCR reaction should be started according to the PCR instrument Setup (see Tab. 4, Tab. 5). 9.3 PCR Instrument Setup Tab. 4: Realtime PCR profile for LightCycler 480II Initial Denaturation 1 min, 95 C Cycles PCR Denaturation Annealing/Extension 45 Cycles 10 sec, 95 C 15 sec, 60 C Temperature Transition Rate / Ramp Rate Maximum Note: Annealing and Extension occur in the same step. 8 RIDA GENE Bacterial Stool Panel I

9 Tab. 5: Realtime PCR profile for Mx3005P Initial Denaturation Cycles PCR Denaturation Annealing/Extension 1 min, 95 C 45 Cycles 15 sec, 95 C 30 sec, 60 C Temperature Transition Rate / Ramp Rate Maximum Note: Annealing and Extension occur in the same step. 9.4 Detection Channel Setup Tab. 6: Selection of appropriate detection channels Realtime PCR instrument Roche LightCycler 480II Agilent Techn. Mx3005P Detection Detection Channel stx1/stx2 440/488 Salmonella spp. 465/510 ICD 533/580 EIEC/Shigella spp. 533/610 Campylobacter spp. 618/660 stx1/stx2 Salmonella spp. ICD EIEC/Shigella spp. ATTO FAM HEX ROX Note RIDA GENE Color Compensation Kit IV (PG0004) is required Check that reference dye is none Campylobacter spp. Cy5 * Due to variations between different cyclers, it may be required to individually adapt the Manual Thres. Fluor Units for channel 1. RIDA GENE Bacterial Stool Panel I

10 10. Quality Control The analysis of the samples is done by the software of the used realtime PCR instrument according to the manufacturer` s instructions. Positive and negative controls have to show correct results (see Table 7, Fig. 1, Fig. 2, Fig. 3, Fig. 4) in order to determine a VALID run. The positive control has a concentration of 10 3 copies/µl. In each PCR run it is used in a total amount of 5 x 10 3 copies, respectively. Tab. 7: For a VALID run, the following conditions must be met: Sample Assay result ICD Ct Target gene Ct PTC Positiv NA * 1 See Quality Assurance Certificate NTC Negativ Ct > 20 0 * 1 No Ct value is required for the ICD to make a positive call for the positive control. If the Positive Control (PTC) is not positive within the specified Ct range but the Negative Control is valid, prepare all new reactions using remaining purified nucleic acids and a new Positive Control. If the Negative Control (NTC) is not negative but the Positive control is valid prepare all new reactions using remaining purified nucleic acids and a new Negative Control. If the required criteria are not met, following items have to be checked before repeating the test: Expiry of the used reagents Functionality of the used instrumentation Correct performance of the test procedure 10 RIDA GENE Bacterial Stool Panel I

11 Fig. 1: Correct run of the positive and negative control (STEC) on the LightCycler 480II Fig. 2: Correct run of the positive and negative control (Salmonella spp.) on the LightCycler 480II RIDA GENE Bacterial Stool Panel I

12 Fig. 3: Correct run of the positive and negative control (EIEC/Shigella spp.) on the LightCycler 480II Fig. 4: Correct run of the positive and negative control (Campylobacter spp.) on the LightCycler 480II 12 RIDA GENE Bacterial Stool Panel I

13 11. Result interpretation The result interpretation is done according to Table 8. Tab. 8: Sample interpretation Target Genes STEC Salmonella spp. EIEC/Shigella spp. Campylobacter spp. ICD Result positive negative negative negative positive/negative STEC detected negative positive negative negative positive/negative negative negative positive negative positive/negative negative negative negative positive positive/negative positive positive negative negative positive/negative positive negative positive negative positive/negative positive negative negative positive positive/negative positive positive positive negative positive/negative positive negative positive positive positive/negative positive positive negative positive positive/negative negative positive positive negative positive/negative negative positive negative positive positive/negative negative positive positive positive positive/negative negative negative positive positive positive/negative positive positive positive positive positive/negative negative negative negative negative positive Salmonella spp. detected EIEC/Shigella spp. detected Campylobacter spp. detected STEC, Salmonella spp. detected STEC, EIEC/Shigella spp. detected STEC, Campylobacter spp. detected STEC, Salmonella spp., EIEC/Shigella spp. detected STEC, EIEC/Shigella spp., Campylobacter spp. detected STEC, Salmonella spp., Campylobacter spp. detected Salmonella spp., EIEC/Shigella spp. detected Salmonella spp., Campylobacter spp. detected Salmonella spp., EIEC/Shigella spp., Campylobacter spp. detected EIEC/Shigella spp., Campylobacter spp. detected STEC, Salmonella spp., EIEC/Shigella spp., Campylobacter spp. detected Negative Target genes not detected negative negative negative negative negative invalid RIDA GENE Bacterial Stool Panel I

14 A sample is evaluated negative, if the sample shows no amplification signal in the detection system, but the Internal Control DNA (ICD) is positive. An inhibition of the PCR reaction or a failure in the extraction procedure can be excluded by the detection of the Internal Control DNA (ICD). A sample is evaluated positive, if both, the sample and the Internal Control DNA, (ICD) show an amplification signal in the detection system. A sample is evaluated positive, if the sample shows an amplification signal in the detection system, but the Internal Control DNA (ICD) is negative. The detection of the internal amplification control is not necessary, because high concentrations of the amplicon can cause a weak or absent signal of the internal amplification control. A sample is evaluated invalid, if both, the sample and the Internal Control DNA (ICD) show no amplification signal in the detection system. The sample contained a PCR inhibitor or a failure occurred in the extraction procedure. The extracted sample needs to be further diluted with PCR water (1:10) and reamplified, or the isolation and purification of the sample has to be improved. 12. Limitations of the method 1. The result of molecular analysis should not lead to the diagnosis, but always be considered in the context of medical history and symptoms of the patient. 2. This assay is only validated for stool samples. 3. Inappropriate specimen collection, transport, storage and processing or a pathogen load in the specimen below the analytical sensitivity can result in false negative results. 4. The presence of PCR inhibitors may cause invalid results. 5. Mutations or polymorphisms in primer or probe binding regions may affect detection of new variants resulting in a false negative result with the RIDA GENE Bacterial Stool Panel I assay. 6. As with all PCR based in vitro diagnostic tests, extremely low levels of target below the limit of detection (LoD) may be detected, but results may not be reproducible. 7. A positive test result does not necessarily indicate the presence of viable organisms. However, a positive result is indicative for the presence of the respective target genes. 14 RIDA GENE Bacterial Stool Panel I

15 13. Test characteristics 13.1 Analytical sensitivity The RIDA GENE Bacterial Stool Panel I multiplex realtime PCR has a detection limit of 10 DNA copies per reaction for STEC, Salmonella spp., EIEC/Shigella spp. and Campylobacter spp. (see Fig. 5, Fig. 6, Fig. 7, Fig. 8). Fig. 5: Dilution series STEC ( DNA copies per µl) on the LightCycler 480II Fig. 6: Dilution series Salmonella spp. ( DNA copies per µl) on the LightCycler 480II RIDA GENE Bacterial Stool Panel I

16 Fig. 7: Dilution series EIEC/Shigella spp. ( DNA copies per µl) on the LightCycler 480II Fig. 8: Dilution series Campylobacter spp. ( DNA copies per µl) on the LightCycler 480II The detection limit of the whole procedure depends on the sample matrix, DNA extraction and DNA concentration. 16 RIDA GENE Bacterial Stool Panel I

17 13.2 Analytical specificity The analytical specificity of the RIDA GENE Bacterial Stool Panel I multiplex realtime PCR is specific for STEC, Salmonella spp., EIEC/Shigella spp. and Campylobacter spp. No crossreaction could be detected for the following species (see Tab. 12): Tab.12: Crossreactivity testing Adenovirus Citrobacter freundii Adenovirus 40, Human, Strain Dugan Adenovirus 41, Human, Strain Tak Clostridium bifermentans Clostridium difficile Entamoeba histolytica Enterobacter cloacae Enterococcus faecalis Serratia liquefaciens Staphylococcus aureus Aeromonas hydrophila Arcobacter butzleri Clostridium perfringens Clostridium sporogenes Astrovirus Clostridium septicum Bacillus cereus Clostridium novyi Giardia lamblia Giardia intestinalis Portland 1 Giardia intestinalis WB Clone C6 Klebsiella oxytoca Bacteroides fragilis Clostridium sordellii Norovirus GG I Staphylococcus epidermidis Vibrio parahaemolyticus Yersinia enterocolitica Campylobacter coli Campylobacter jejuni Cryptosporidium muris Cryptosporidium parvum Norovirus GG II Proteus vulgaris Campylobacter lari subsp. Lari E. coli (O26:H) Pseudomonas aeruginosa Candida albicans E. coli (O6) Rotavirus Adenovirus 1, Human, strain Adenoid 71 Adenovirus 7, Human, Strain Gomen RIDA GENE Bacterial Stool Panel I

18 13.3 Analytical reactivity The reactivity of the RIDA GENE Bacterial Stool Panel I multiplex realtime PCR was evaluated against multiple stx1/stx2 subtypes, Campylobacter species, Salmonella serotypes and EIEC/Shigella spp. (see Tab. 13). All STEC genogroups, Campylobacter species, Salmonella serotypes and EIEC/Shigella spp. of the panel were detected by the RIDA GENE Bacterial Stool Panel I multiplex realtime PCR. Tab.13: Analytical reactivity testing stx1subtypes stx1a + stx1c + stx1d + stx2subtypes stx2a + stx2d + stx2g + stx2b + stx2e + stx2c + stx2f + Campylobacter species C. coli + C. jejuni + C. lari + Salmonella serotypes S. enteritidis + S. typhimurium + S. heidelberg + S. brandenburg + S. agona + S. infantis + S. kiel + S. paratyphi A + S. gloucester + S. haifa + S. wilhelmsburg + S. essen + S. virchow + S. montevideo + S. blegdam + S. rostock + S. moscow + S. pullorum + S. wernigerode + S. hadar + S. duesseldorf + S. glostrup + S. poona + S. oranienburg + S. senftenberg + S. bongori + S. bovismorbificans + S. derby + S. anatum + S. newport + S. goldcoast + S. dublin + S. livingston + S. muenchen + S. kentucky + S. amsterdam + S. berta + S. caracas + S. ealing + S. augustenbourg + S. arizonae + S. schwarzengrund + S. nottingham + S. ohio + Shigella S. flexneri + 18 RIDA GENE Bacterial Stool Panel I

19 Literature 1. World Gastroenterology Organisation Global Guidelines: Acute diarrhea in adults and children : a global perspective. 2. UNICEF/WHO, Diarrhoea: Why children are still dying and what can be done, FDA Bad Bug Book 2nd Edition. Foodborne Pathogenic Microorganisms and Natural Toxins Handbook. 4. RuizPalacios GM. Clinical Infectious Diseases 2007; 44: CDC. National Salmonella Surveillance Overview. Atlanta, Georgia: US Department of Health and Human Services, CDC, Majowicz SE et al. Clinical Infectious Diseases 2010; 50: Pui CF et al. International Food Research Journal 2011; 18: RIDA GENE Bacterial Stool Panel I